Genetic engineering techniques are frequently used in the production of physiologically active polypeptides. Basically, genetic codes are normally very faithfully translated, but sometimes an amino acid or an amino acid derivative which does not correspond to the codon table is incorporated into the polypeptides during translation. For example, the percentage of ribosomal mRNA mistranslation was 10−4 per codon from the experiment of [35S]Cys incorporation into highly purified cysteine-free E. coli flagellin protein. However, probability of cysteine incorporation into this protein greatly increases in the presence of an antibiotic streptomycin; this is probably because Cys codons (UGU and UGC) are mistranslated for Arg codons (CGU and CGC) (Voet and Voet, Biochemistry, Vol. 2, second edition, Tokyo Kagaku Dojin. pp. 869-870, 1998).
Tsai et al. reported that norleucine is incorporated with high frequency into the location that should naturally be occupied by methionine in the expression of human IL-2 in E. coli. (Tsai, L. B. et al., Biochem. Biophys. Res. Commun., Vol. 156, pp. 733-739, 1988). A similar report is found in the expression of bovine somatotropin (Bogosian, G. et al., J. Biol. Chem., Vol. 264, pp. 531-539, 1989). In both cases, the authors assume that norleucine was synthesized in cells by the activation of the leucine synthetic pathway in E. coli and added to methionine tRNA in place of methionine, and thus incorporated into the expressed proteins.
In addition to misincorporation of norleucine, Apostol et al. shows that norvaline was misincorporated into the location that should be occupied by leucine in the production of recombinant hemoglobin by E. coli (Apostol I. et al., J. Biol. Chem., Vol. 272, pp. 28980-28988, 1997). In this case, the authors also assume that the activation of the leucine synthetic pathway in E. coli led to the production of norvaline, which was then incorporated in place of leucine.
As for the above case in which norleucine is incorporated into intended polypeptides in place of methionine, a method for reducing the incorporation of norleucine in heterologous polypeptides expressed in transformed microorganisms grown in a medium by increasing the concentration of methionine and/or leucine or decreasing the amount of norleucine in the fermentation medium or combining both has been known (Japanese Patent No. 2879063, U.S. Pat. No. 5,599,690).
The present inventors investigated a method for efficiently producing human atrial natriuretic peptide (hereinafter also referred to as hANP, the amino acid sequence shown in SEQ ID NO: 1; Kangawa, K. et al., Biochem. Biophys. Res. Commun., Vol. 118, pp. 131-139, 1984) by genetic engineering using E. coli as a host cell, and succeeded in constructing a method for efficiently producing hANP from a fusion protein (Japanese Patent No. 1963624). In this method, the fusion protein comprises a protective peptide consisting of the N-terminal 97 amino acids of E. coli β-galactosidase, a linker sequence of 3 amino acid residues including a lysine residue (Gln-Phe-Lys) and hANP, and the gene for this fusion protein is encoded on a pBR322-derived expression vector. Transcription of the fusion protein gene is controlled by an E. coli-derived lactose promoter, and the expressed fusion protein accumulates as inclusion bodies in E. coli. The resulting fusion protein is solubilized by a denaturing agent and then treated with a protease specifically recognizing and cleaving the lysine residue, API (Achromobacter protease I [Masaki, T. et. al., Biochim. Biophys. Acta. Vol. 660, pp. 51-55, 1981]) to release hANP, which is purified by chromatography to give a final product hANP.
During studies of this hANP production process, the present inventors found an impurity that has similar physiochemical properties to those of hANP and cannot be easily separated by chromatography. This impurity is detected as a substance eluting slightly after hANP in analytical reverse phase high-pressure liquid chromatography (RP-HPLC) and exists in a proportion of about 5% to hANP cleaved by enzymatic reaction (this impurity byproduct polypeptide will be hereinafter referred to as R1). This R1 was hard to separate as its elution peak overlapped the tail portion of hANP elution curve in preparative HPLC used on a production scale.
As hANP is used as a medicine for treating acute heart failure, it is important to provide hANP with high purity for such a medical use. Current production processes sufficiently ensure the medical level of purity, but have a problem in production costs because the byproduct polypeptide R1 must be removed during the purification step at the expense of a yield loss. Thus, it has been an important challenge to find a means for reducing the formation of the impurity in the production of high-purity hANP.
In the production of recombinant polypeptides, impure byproducts must be removed during the purification step at the expense of a yield loss leading to a possible problem in production costs, and it is important to find a means for reducing the formation of the byproducts in the production of high-purity recombinant polypeptides. Therefore, an object of the present invention is to provide a method for reducing the formation of byproducts in the production of a polypeptide and a method for producing a recombinant polypeptide characterized by reducing the formation of byproducts.